Endothelial cell permeability assay
Endothelial cell permeability assay
Changes in endothelial barrier function regulate vascular leakage into tissues to maintain homeostasis. Altered endothelial cell permeability is important in the pathophysiologic process of many diseases, and a series of in vivo and ex vivo endothelial cell barrier permeability assays have been established.
Principle
Evans Blue is a commonly used azo dye that has a high affinity for plasma albumin and can be used to label albumin. Under physiologic conditions, the endothelium is impermeable to albumin; whereas, under pathological conditions, dysfunction of endothelial intercellular junction proteins leads to increased barrier permeability, at which point Evans blue-albumin leakage can be detected in a monolayer endothelial cell model.
Operation method
Permeability assay of the endothelial cell barrier
Principle
Evans Blue is a commonly used azo dye that has a high affinity for plasma albumin and can be used to label albumin. Under physiologic conditions, the endothelium is impermeable to albumin; whereas, under pathological conditions, dysfunction of endothelial intercellular junction proteins leads to increased barrier permeability, at which point Evans blue-albumin leakage can be detected in a monolayer endothelial cell model.
Materials and Instruments
Transwell cell chambers, cell culture plates, cell culture medium, transmembrane cell resistivity meter, Evans Blue (EB), Bovine Serum Albumin (BSA). Move 1. Monolayer cell barrier establishment: Cells were digested, counted and the cell concentration was adjusted to 1×105 cells/mL, 1 mL of cell suspension was pipetted and inoculated into a Transwell with a pore size of 3 μm and inserted into a 24-well cell culture plate, 1 mL of complete cell culture medium was added in advance to the lower chamber of the plate, and the plate was placed in a cell culture incubator at 37 ℃ with 5% CO2, and transmembrane resistance (TEER) was measured by a cytoresistivity meter and the value was recorded until the TEER was stable and continuous, which was considered as the formation of a monolayer of cells. The transmembrane electrical resistance (TEER) was measured every two days using a cytoresistivity meter and the value was recorded, and the monolayer cell barrier was considered to be formed when the TEER was stable and sustained; 2. Evans Blue-BSA working solution was prepared: 2% EB storage solution was mixed with 60 times the volume of 4% BSA, and diluted to a final concentration of 0.67 mg/mL with PBS; 3. Evans blue leakage assay: Remove the Transwell, wash with PBS twice, put into a new 24-well plate, add 600 μL of 4% BSA solution to the lower chamber, and add 100 μL of EB-BSA working solution to the upper chamber to ensure that the height of the upper chamber is the same as the liquid level of the lower chamber and to eliminate liquid leakage caused by the difference in liquid level height, and equilibrate at 37 ℃, 5% CO2 cell culture incubator for 1 h. The cell culture incubator was then incubated for 1 h. The cell culture incubator was then incubated for 1 h. The liquid level was then washed with PBS and the liquid washed with PBS; 4. Permeability assessment: ① Remove the Transwell, aspirate the liquid from the lower chamber into a 1.5 mL EP tube, centrifuge at 1,000 rpm for 5 min, and add 200 μL to a 96-well plate; ② Dilute the EB-BSA working solution (the initial concentration of 0.67 mg/mL) in multiple folds, and add 200 μL to a 96-well plate, and measure the OD value and the OD value with a multifunctional enzyme labeling instrument at 620 nm. Measure the OD value at 620 nm using a multifunctional enzyme labeling instrument and make a standard curve; ③ Calculate the concentration of EB in the liquid in the lower chamber of the Transwell according to the standard curve, which represents the amount of Evans blue leaking from the upper chamber to the lower chamber, i.e., the permeability of monolayers of endothelial cells. Caveat 1. According to the experimental needs, different pore sizes of Transwell can be selected, commonly used 0.4 μm~3 μm pore sizes;2. The integrity of the barrier should be confirmed before modeling, and the formation of an intact endothelial barrier can be assessed by TEER;3. When detecting the leakage of EB-BSA, it is necessary to ensure that the liquid level of the upper and lower Transwells are the same to avoid leakage caused by the difference in liquid height. Common Problems 1. What is the recommended cell density for inoculation? Different experiments use different kinds of cells, and the culture time and method are also different, we suggest to refer to the literature. 2. Is it necessary to coat the cell membrane, e.g. collagen coating? Generally speaking, if the cells are well adherent to the wall when cultured on the TC-treated surface of the culture vessel, no additional coating of the cell membrane is required. Only if the cells are not well adherent and have required coating in previous cultures is it necessary to coat the cell membranes, but please be aware that the coating layer may interfere with the subsequent exchange of substances, and it is recommended to refer to the relevant literature. 3. Endothelial cells grow unevenly in the Transwell. To optimize cell growth on the Transwell, avoid inoculation of culture flasks with more than 85%~90% cell coverage at the bottom; after cell inoculation is complete, be sure to carefully transfer the Transwell plate to the incubator. Avoid leaking culture medium containing suspended cells from the Transwell into the culture plate. 4. 4. Unstable TEER values. Equilibrate the chambers to room temperature before performing TEER measurements on monolayer endothelial cell models to obtain more uniform results. TEER values vary between endothelial cell plateaus; note that the volume of liquid in the upper and lower chambers affects the measurement values, so strictly standardize the working volume for each measurement. For more product details, please visit Aladdin Scientific website.